Post on 08-Oct-2020
27-11-2014
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Phagetherapy as alternative to antibiotictherapy in animal production
Joana AzeredoCentre of Biological EngineeringUniversity of Minho
www.ceb.uminho.pt/bbig
Symposium zum Europäischen Antibiotikatag 2014 (18th Nov 2014)
Presentation Outline
�Bacteriophages as biocontrol agents
�Advantages and disadvantages of phage therapy compared to antibiotherapy
�Examples: Phage therapy in poultry industry to control
� Salmonella
� Campylobacter
� E. coli
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Bacteriophages as biocontrol agents
� Bacteriophages (phage) are viruses that infect bacteria
� Phages are ubiquitous in the environment
� Phage can be highly specific or have a broad host range
� Phage specifically recognize and bind to their host through specific receptors
� Bacteriophages can be virulent (lytic) or temperate
T4- like bacteriophage
Only lytic phages have effective biocontrol properties
108 phage particles
Highlights in the development of phage as a potential therapeutic agent for bacterial infections (Levin and Bull, Nature, 2004)
Antibiotics surpassed phage
therapy
Renewed enthusiasm on account of
antibiotic resistance
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Bacteriophages as biocontrol agents
2005 2006 2007 2013
US Environmental Protection Agency approved AgriphageTM
FDA approved phage-based preparations for E. coli and Salmonella
control on RTE EcoShieldTM, SalmoFresh™
FDA approved
ListShieldTM
from Intralytix
and Listex TM
from MICREOS
FDA & USDA approve SALMONELEX™ as processing-aid against Salmonella
Discovery of bactriophages
d´Herelle and Twort
bacteriophages were for the
first time considered
generally recognized as safe
(GRAS)
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5
Bacteriophages as biocontrol agents
2005 2006 2007 2013
US Environmental Protection Agency approved AgriphageTM
FDA approved phage-based preparations for E. coli and Salmonella
control on RTE EcoShieldTM, SalmoFresh™
FDA approved
ListShieldTM
from Intralytix
and Listex TM
from MICREOS
FDA & USDA approve SALMONELEX™ as processing-aid against Salmonella
Approved phage products
Food additives
Poultry Productions
Human therapy
Bacteriophages as biocontrol agents
ADVANTAGES
OVER ANTIBIOTICSPHAGES
Highly specific
Do not disturb
normal flora
Ubiquitous
No
environmental
impact
Innocuous
Not toxic to
animals and
plants
Effective against
bacteria resistant
to antibiotics
Bind to bacterial
antigens/
(capsules, LPS)
Resistance strains
usually less virulent
Self replicating
Increase in
time
Lower doses
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Bacteriophages as biocontrol agents
DISADVANTAGES
OVER ANTIBIOTICSPHAGES
Highly specific
Do not disturb
normal flora
Ubiquitous
No
environmental
impact
Innocuous
Not toxic to
animals and
plants
Effective against
bacteria resistant
to antibiotics
Bind to bacterial
antigens/
(capsules, LPS)
Resistance strains
usually less virulent
Self replicating
Increase in
time
Lower doses
Bacteriophages as biocontrol agents
DISADVANTAGES
OVER ANTIBIOTICSPHAGES
Highly specific
Bind to bacterial
antigens/
(capsules, LPS)
Development of phage resistance Low efficacy
Low efficacy
Use of a cocktail
of different
phages
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Phage therapy in animal production: Poultry industry
High incidence of case reported outbreaks of Salmonella and Campylobacter
Salmonella
Campylobacter
E .coli
• High mortality rate
• Carcass rejection at slaughter
• Low feed conversion rate
• Weight loss
Represents very important economical losses in poultry industry
• High treatment cost
Avian Colibacillosis
N=214,286
N=91,034
Phage therapy in animal production: Poultry industry
Isolation of phages
Selection of phages
Phage characteriza
tion
Large scale phage
production
In vivo phage
efficacy trials
Isolation of phages
• Waste water
• Effluents
• Animal
• Poultry houses
Phages
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Phage therapy in animal production: Poultry industry
Isolation of phages
Selection of phages that
Phage characteriza
tion
Large scale phage
production
In vivo phage
efficacy trials
Selection of phages
A cocktail of phages with:
- Broad lytic spectra
- Complementary lytic spectra
- Cross resistances
- Lytic performance
Low High
Phage therapy in animal production: Poultry industry
Isolation of phages
Selection of phages
Phage characteriza
tion
Large scale phage
production
In vivo phage
efficacy trials
Phage characteriza
tion
RFLP analysis Phage genome sequencingTEM observation
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Phage therapy in animal production: Poultry industry
Isolation of phages
Selection of phages
Phage characteriza
tion
Large scale phage
production
In vivo phage
efficacy trials
Large scale phage
production
Tissue culture plates
2-5 L fermenters
In vivo phage
efficacy trials
Confined rooms Farms
Phage therapy in animal production: Campylobacter Control
Genome sequence:
Unique genes
Similarity to T4-like phages
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Phage therapy in animal production: Campylobacter Control
2
3
4
5
6
7
0 1 2 3 4 5 6 7
Nu
mb
er
of
C.c
oli
(lo
g 1
0 c
fu/g
)
Days post treatment
negative control
phage - oral gavage
phage - feed
Numbers of C.jejuni 2140CD1 in faeces from broilers orally
administered a phage cocktail by gavage (Carvalho et al., 2010)
Results of animal trials
Average 2-log reduction in Campylobacter
caecal content after 2 days of treatment
single dose by oral gavage
single dose incorporated in food
Phage cocktail incorporated in the birds’
feed led to an earlier reduction in
Campylobacter titer (2 days post-phage
administration)
Reduction of approximately 2 log cfu/g is
considered, by mathematical models to lead
to a 30-fold reduction in the incidence of
campylobacteriosis associated with
consumption of chicken meals.
In vivo acquisition of phage resistance
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Chickens groupCampylobacter titre
(log10cfu/g)
Resistant
strains
(%)
Non treated group 6.50 6
Treated group 4.29 13
Bacteria can naturally acquire phage resistance
�The resistant phenotype was not hindering the
ability of the Campylobacter to colonize the
chickens
�The majority of phage resistant strains reverted
to sensitive phenotypes
Phage therapy in animal production: Campylobacter Control
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Phage therapy in animal production: Salmonella Control
Salmonella Tiphimurium NCTC 12416 - subsp. I +
Salmonella NCTC 13349 - subsp. I +
Salmonella spp. SGSC 3047 - subsp. II +
Salmonella spp. SGSC 3039 - subsp. II +
Salmonella Arizonae 3063 - IIIa +/-
Salmonella Arizonae 83 - IIIa -
Salmonella spp. SGSC 3069 - subsp. IIIb +
Salmonella spp. SGSC 3068 - subsp. IIIb +
Salmonella spp. SGSC 3086 - subsp. IV +/-
Salmonella spp. SGSC 3074 - subsp. IV +
Salmonella Bongori SGSC 3103 - subsp. V +
Salmonella Bongori SGSC 3100 - subsp. V +
Salmonella spp. SGSC 3118 - subsp. VI +
Salmonella spp. SGSC 3116 - subsp. VI +
Salmonella spp. SGSC 3121 - subsp. VII +
Salmonella spp. SGSC 3120 - subsp. VII +
Escherichia coli n9 +/-
Escherichia coli n5 +/-
Escherichia coli CECT 434 (ATCC 25922) +/-
Escherichia coli BL21 +
Escherichia coli K12 +
Produce the phage in non-
pathogenic E. coli
Phage therapy in animal production: Salmonella Control
Salmonella caecal content (CFU/g) after 1 and 2 days post
administration of PVP-SE1 by oral gavage (PhageVet-P
report)
3 log reduction of
the treated group
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Phage therapy in animal production: APEC Control
Relative comparison (%) of the isolated strains according to susceptibility, intermediate susceptibility or resistance to a range ofantibiotics commonly used for therapy in poultry industry. AMP -Ampicillin; DO - Doxycycline; ENR - Enrofloxacin, STX -Sulphamethoxazole / Trimethropim, NA - Nalidixic acid, PIP - Pipemidicacid, TE - Tetracycline, OA - Oxolinic acid; AML- AmoxycillinOlveira et al., 2010
920 APEC isolated from 2003-2010 in Portugal are
highly resistant to antibiotics, over 80% are
resistant to tetracycline
Phage therapy in animal production: Avian Pathogenic Control
(A)phi F78E (Myoviridae) (B) phi F258E (Siphoviridae) (C) phi
F61E (Myoviridae)
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Phage therapy in animal production: Avian Pathogenic Control
Phage administration resulted in a decrease,
in average, of 25.0 % on chickens’ mortality
and of 41.7 % on morbidity
Phage administration resulted in a decrease,
in average, of 25.0 % on chickens’ mortality
and of 41.7 % on morbidityScore of injuries, mortality and morbidity in the treated and untreated group (Oliveira et al., 2010)
0
20
40
60
80
100
Morbidity Mortality
%
0
1
2
3
4
5
Pathology score
Scor
e
phage treated group
untreated group
Phage therapy in animal production: Avian Pathogenic Control
LARGE SCALE ANIMAL EXPERIMENTS5x107PFU/ml, administered in a single application, orally and by spray. In 11 flocks
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Conclusions
� Phages offer a great potential in controlling
bacterial pathogens in live animals
Facts and Challenges
Facts Challenges
Conclusions
� Phages offer a great potential in controlling
bacterial pathogens in live animals
Facts and Challenges
Facts Challenges
� Most phages are sensitive to low pH Neutralization of the pH
Phage encapsulation
� Phage resistant phenotypes
Phage application prior to slaughter
Environmental decontamination
� Phages are highly specific so the phage
cocktail should be designed are updated for
each particular application
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Conclusions Facts and Challenges
� Phages are highly specific so the phage
cocktail should be designed are updated for
each particular application
Animal Production Facilities
• Isolation of the pathogenic bacteria
Laboratory
• Selection of the most effective phage cocktail
Animal production
• Phage administration
Phage Library of well
characterized phages
Conclusions
� Phages offer a great potential in controlling
bacterial pathogens in live animals
Facts and Challenges
Facts Challenges
� Phages are sensitive to low pH Neutralization of the pH
Phage encapsulation
� Phage resistant phenotypes
Phage application prior to slaughter
Environmental decontamination
� Phages are highly specific so the phage
cocktail should be designed are updated for
each particular application What would be the regulatory frame for
this type of product?
Are pharma companies interested in
investing ?
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Acknowledgments
THANK YOU!
Ana Oliveira
Carla Carvalho
Sílvio Santos
Sanna Sillankorva
•Rob Lavigne (KULeuven)
•Ben Ganon (University of Bristol)
•Ian Sutherland (Edinburgh University)
•Victor Krylov (State Institute for Genetics of Industrial
Microorganisms)
•Hans Ackermann (Laval University)
•Andrew Kropinski (University of Guelph)
External collaborators